Cationic lipid compound as well as preparation method and application thereof

Abstract

The invention discloses a cationic lipid compound as well as a preparation method and application thereof, and the cationic lipid compound has the following structural characteristics: 1, the lipid compound has a multi-amino amidino head structure, so that the delivery efficiency of nucleic acid molecules can be improved; 2, the lipid compound has a phenylamidine structure, so that the structural stability of amidino can be improved, and the physical and chemical stability of lipid molecules can be improved; and 3, double biodegradation structures based on amidino and amidino bonds on two sides of a benzene ring in a lipid compound structure can be hydrolyzed and broken into small molecules after nucleic acid molecules are effectively delivered, so that the cell biocompatibility of lipid molecules can be improved, and the in-vivo safety can be improved. In conclusion, the multi-amino amidino cationic lipid molecule has the characteristics of high delivery efficiency, good cell biocompatibility and excellent storage stability, provides more choices for nucleic acid drug delivery, and has important significance for development and application of nucleic acid drugs.

Description

Technical Field

[0001] This invention belongs to the field of lipid materials technology, specifically relating to a cationic lipid compound, its preparation method, and its application. Background Technology

[0002] Nucleic acid molecular therapy is an emerging and promising treatment strategy. It is precisely defined as a method that uses nucleic acid molecules as drugs to treat diseases by altering gene expression levels or regulating cellular function. Nucleic acid molecular drugs include DNA and RNA (mRNA, siRNA, microRNA, etc.). The advantages of nucleic acid molecular therapy are: 1. High specificity, enabling personalized treatment; 2. Direct action on genetic information to regulate gene expression, providing a new strategy for diseases that are difficult to cure with traditional treatments; 3. Overcoming the limitation of traditional drugs being unsuitable for drug production. However, a major challenge facing nucleic acid molecular therapy is achieving effective intracellular delivery. Both RNA and DNA are negatively charged macromolecules, resulting in poor membrane penetration. Furthermore, nucleic acid molecules are easily degraded by nucleases abundant in the body, making it difficult for nucleic acid molecular drugs to enter cells and exert their therapeutic effects. Therefore, to fully realize the therapeutic potential of nucleic acid molecular therapy, a reliable nucleic acid molecular delivery system is needed to achieve effective intracellular delivery.

[0003] Lipid nanoparticles (LNPs) are among the most advanced nucleic acid delivery systems available today, exhibiting superior delivery efficiency and safety. Existing LNPs primarily consist of four classic components: 1. Ionizable cationic lipids, which effectively encapsulate nucleic acid molecules and facilitate efficient endosome escape; 2. Cholesterol, which mainly enhances the stability of the lipid nanoparticles; 3. Helper lipids, which primarily promote endocytosis of the nanoparticles; and 4. PEGylated lipids, which primarily prolong the plasma circulation time of the nanoparticles and improve their overall stability. Among these four components, the ionizable cationic lipid molecules are the most crucial. Their role is to maintain a positive charge in an acidic environment, effectively encapsulating negatively charged nucleic acid molecules through electrostatic adsorption. The nucleic acid-encapsulated lipid nanoparticles maintain near-electroneutrality in plasma. However, upon entering cells, the cationic lipid components re-acquire a positive charge in the acidic endosome environment, disrupting the negatively charged endosome membrane and releasing the nucleic acid molecules to exert their therapeutic effect. However, existing commercially available and developed lipid nanoparticles all suffer from one or more of the following problems: (1) low encapsulation efficiency of nucleic acid molecules; (2) low efficiency of intracellular delivery and endosome escape of nucleic acid molecules; (3) high cytotoxicity of lipid nanoparticles and poor safety of in vivo delivery; (4) poor stability of lipid nanoparticles. Therefore, it is necessary to provide a new lipid nanoparticle that combines high delivery efficiency, high encapsulation efficiency, good cell biocompatibility, and excellent stability. Summary of the Invention

[0004] To overcome the problems existing in the prior art, one objective of the present invention is to provide a cationic lipid compound. A second objective of the present invention is to provide a method for preparing the above-mentioned cationic lipid compound. A third objective of the present invention is to provide a lipid composition. A fourth objective of the present invention is to provide applications of the above-mentioned cationic lipid compound and lipid composition.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a cationic lipid compound with the structural formula shown in Formula I: Formula I; Wherein, m, n, and p are independently selected from integers 1~8, 0~8, and 1~8, respectively; The X 1 It is an amide bond or an ester bond; The R 1The optional substituted (C1-C6) alkylamine group, the optional substituted bis(C1-C6) alkylamine group, the optional substituted aromatic amine group, or the optional substituted nitrogen heterocyclic group; wherein the nitrogen atom of the optionally substituted nitrogen heterocyclic group is... Connected; The R 2 Selected from (C5-C) which are optionally substituted 25 )alkyl, optionally substituted (C5-C 25 alkenyl; The R 3 Selected from halogen, cyano, isocyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 carbocyclic, C6-C 10 Aryl; k is an integer selected from 0 to 4; when k ≥ 2 are different, multiple R 3 They can be the same or different; The substitution refers to the presence of a substituent at one or more substituted positions of the group, wherein the substituent is independently halogen, cyano, isocyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 carbocyclic, C6-C 10 Aryl, C5-C 10 Mixed aromatic compounds.

[0006] The term "substituted" means that the alkyl, alkenyl, alkynyl, carbocyclic, heterocyclic, aryl, and heteroaryl groups as defined in this application are optionally substituted. "Optionally substituted" means either independently unsubstituted or substituted by one or more substituents (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" carbocyclic, "substituted" or "unsubstituted" heterocyclic, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl). Generally, the term "substituted" means that at least one hydrogen atom present on the group is replaced by a permissible substituent, such as a substituent that, upon substitution, produces a stable compound, for example, a compound that does not spontaneously undergo transformations such as by rearrangement, cyclization, elimination, or other reactions.

[0007] Preferably, its structural formula is as shown in Formula II or Formula III: Formula II; Formula III; Preferably, m is selected from 1, 2 or 3.

[0008] Preferably, n is selected from 0, 1, or 2.

[0009] Preferably, p is selected from 1, 2, or 3.

[0010] Preferably, the X 1 Selected from: , , , The dashed lines represent the connection points of the functional groups. The left side of each depicted structure is... Combine; The R 3 Independently selected from hydrogen or optionally substituted (C1-C6) alkyl groups.

[0011] Preferably, the R 1 Selected from methylamino, ethylamino, propylamino, dimethylamino, diethylamino, dipropylamino, aniline, naphthylamino, anthraceneamino, pyridyl, and pyrrole.

[0012] Preferably, the R 2 Selected from (C5-C) which are optionally substituted 15 )alkyl, optionally substituted (C5-C 15 The substituent is an alkenyl group, wherein the substituent is independently a halogen or a C1-C6 alkyl group.

[0013] More preferably, the R 2 It is a saturated straight-chain hydrocarbon group.

[0014] More preferably, the cationic lipid compound has the structural formula shown in any of the following chemical formulas: , ; In the above structural formula, m is 1 or 2, n is 0, 1 or 2, p is 1 or 2, and z is an integer from 6 to 13.

[0015] The second aspect of the present invention provides a method for preparing the cationic lipid compound described in the first aspect, comprising the following steps: reacting a compound of formula F with a compound of formula A to obtain the compound of formula I; The structural formulas of compound F and compound A are shown below: Formula F; ; Wherein, m, n, p, k, X 1 R 1 R 2 R 3 As stated in the first aspect.

[0016] Preferably, the reaction time is independently 0.5h-3h.

[0017] More preferably, the reaction time is 0.75h-1.2h.

[0018] Preferably, the reaction temperature is independently between 90°C and 130°C.

[0019] More preferably, the reaction temperature is 100℃-120℃.

[0020] Preferably, the molar ratio of compound F to compound A is 1:(2-10).

[0021] More preferably, the molar ratio of compound F to compound A is 1:(3-4).

[0022] Preferably, the reaction in the preparation method of compound I is carried out in the presence of a catalyst.

[0023] More preferably, the catalyst is aluminum chloride.

[0024] More preferably, the molar ratio of the compound of formula F to the catalyst is 1:(1-2).

[0025] More preferably, the molar ratio of the compound of formula F to the catalyst is 1:(1.1-1.3).

[0026] Preferably, the X 1 Selected from: And R 3 When hydrogen is present, the preparation method of compound F includes the following steps: compound E and compound P A The compound is reacted to obtain compound F; Compound E and Compound P A The structural formulas of the compounds are as follows: Formula E; Formula P A .

[0027] More preferably, the reaction in the preparation method of compound F is carried out in the presence of a catalyst, which includes 1-hydroxybenzotriazole (HOBt), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N,N-diisopropylethylamine (DIEA).

[0028] More preferably, in the method for preparing compound F, compound E and compound P... A The molar ratio of the compounds is 1:(1-2).

[0029] More preferably, in the method for preparing compound F, the reaction time is 4-6 hours.

[0030] More preferably, the method for preparing the compound of formula E includes the following steps: reacting the compound of formula C with the compound of formula D to obtain the compound of formula F; The structural formulas of compounds C and D are as follows: Formula C; Formula D.

[0031] More preferably, in the method for preparing compound E, the molar ratio of compound C to compound D is 1:(2-5).

[0032] More preferably, in the method for preparing compound E, the reaction is carried out in the presence of a photoinitiator and under light irradiation.

[0033] A third aspect of the present invention provides a lipid composition comprising the cationic lipid compound described in the first aspect, and further comprising at least one of the following lipids: (i) one or more non-cationic lipids; (ii) One or more cholesterol-based lipids; (iii) One or more PEG-modified lipids.

[0034] Preferably, the lipid composition is microparticles, nanoparticles, liposomes, lipid nanoparticles, or microbubbles.

[0035] The fourth aspect of the present invention provides the use of the cationic lipid compound described in the first aspect, or the lipid composition described in the third aspect, in the delivery of bioactive substances.

[0036] Preferably, the bioactive substance is a small molecule compound, nucleic acid, protein, peptide, metal, isotope-labeled compound, or vaccine.

[0037] More preferably, the nucleic acid is DNA, mRNA, siRNA, or circular RNA.

[0038] Preferably, the application is a drug delivery system, and the delivered drug may be a therapeutic agent, a diagnostic agent, or a preventive agent.

[0039] The drug delivery system may be in particle form. In some embodiments, the particle diameter is in the range of 1 μm to 1000 μm. In some embodiments, the particle diameter is in the range of 1 nm to 1000 nm. When the particle size range is in the range of 1 nm to 1000 nm, it is a nanoparticle as commonly known in the art. The particles may be prepared using any method known in the art. These methods include (but are not limited to) spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, nanoprecipitation, microfluidics, simple and complex agglomeration, and other methods well known to those skilled in the art.

[0040] More preferably, the drug is selected from any one of the following: (1) a drug for treating tumors; (2) a drug for treating autoimmune diseases; (3) a drug for treating infectious diseases; (4) a drug for treating infectious diseases; (5) a drug for treating cardiovascular diseases; (6) a drug for treating diabetes; (7) a drug for treating nervous system diseases; (8) a drug for treating respiratory system diseases; and (9) a drug for treating blood system diseases.

[0041] The beneficial effects of this invention are: This invention provides a cationic lipid compound with the following structural features: 1. The lipid compound has a multi-aminomidine head structure, which can improve the delivery efficiency of nucleic acid molecules; 2. The lipid compound has a benzamide structure, which can increase the structural stability of the amidine group and improve the physicochemical stability of the lipid molecule; 3. The benzene ring in the lipid compound structure has a dual biodegradable structure based on amidine and amide bonds on both sides, which can undergo hydrolysis and break down into smaller molecules after effectively delivering nucleic acid molecules, thereby increasing the cell biocompatibility of the lipid molecule and improving in vivo safety. In summary, the multi-aminomidine cationic lipid molecule of this invention combines high delivery efficiency, good cell biocompatibility, and excellent storage stability, providing more options for nucleic acid drug delivery and having significant implications for the development and application of nucleic acid drugs.

[0042] In addition, this invention develops a two-component LNP formulation based on polyaminoamidine cationic lipid molecules, which is simpler to process than the traditional four-component LNP formulation. Furthermore, performance can be further enhanced by adding a third component (such as a permanent cationic lipid, phospholipid, or cholesterol substitute). Attached Figure Description

[0043] Figure 1 The mass spectrometry characterization results are for A1P1C1T1. Figure 2 The mass spectrometry characterization results are for A1P1C1T2; Figure 3 The mass spectrometry characterization results are for A1P1C1T3; Figure 4 The mass spectrometry characterization results are for A1P1C1T4; Figure 5 The mass spectrometry characterization results are for A1P1C1T5; Figure 6 The mass spectrometry characterization results are for A1P1C1T6; Figure 7 The mass spectrometry characterization results are for A1P1C1T7; Figure 8The mass spectrometry characterization results are for A1P1C2T1; Figure 9 The mass spectrometry characterization results are for A1P1C2T2; Figure 10 The mass spectrometry characterization results are for A1P1C2T3; Figure 11 The mass spectrometry characterization results are for A1P1C2T4; Figure 12 The mass spectrometry characterization results are for A1P1C2T5; Figure 13 The mass spectrometry characterization results are for A1P1C2T6; Figure 14 The mass spectrometry characterization results are for A1P1C2T7; Figure 15 The 1H NMR spectrum of A1P4C2T4; Figure 16 The 1H NMR spectrum of C2T4; Figure 17 The 1H NMR spectrum of P4C2T4; Figure 18 To evaluate the transfection efficiency of 14 polyaminomidine cationic lipid molecules selected in the first stage of screening in 293T cells; Figure 19 The image shows the EGFP mRNA transfection of A1P1C2T3 lipid molecules in 293T cells, where (A) is a bright-field imaging image; (B) is a fluorescence imaging image. Figure 20 To evaluate the transfection efficiency of 24 polyaminomidine cationic lipid molecules selected in the second stage of screening in HT-22 cells; Figure 21 The image shows the EGFP mRNA transfection of A1P1C2T3 lipid molecules in HT-22 cells, where (A) is a bright-field imaging image; and (B) is a fluorescence imaging image. Figure 22 To evaluate the transfection efficiency of 24 polyaminomidine cationic lipid molecules selected in the second stage of screening in BV2 cells; Figure 23 The image shows the EGFP mRNA transfection of A1P1C2T3 lipid molecules in BV2 cells, where (A) is a bright-field imaging image; (B) is a fluorescence imaging image. Figure 24 The results of transfection of 16 polyaminomidine cationic lipid molecules selected in the third stage of screening into HT-22 cells; Figure 25 The results of transfection of 16 polyaminomidine cationic lipid molecules selected in the third stage of screening into BV2 cells; Figure 26Screening for the optimal two-component formulation of A1P1C2T3 in 293T cells; Figure 27 Screening for the optimal transfection dose for A1P1C2T3; Figure 28 Evaluation of the cytotoxicity of A1P1C2T3; Figure 29 For the storage stability evaluation of A1P1C2T3; Figure 30 Transfection images of 293T cells after three weeks of storage using A1P1C2T3, where (A) is a bright-field imaging image; (B) is a fluorescence imaging image. Figure 31 Transfection images of HT-22 cells after three weeks of storage using A1P1C2T3, where (A) is a bright-field imaging image; (B) is a fluorescence imaging image. Figure 32 The 1H NMR spectrum of C2T3; Figure 33 The 1H NMR spectrum of P1C2T3; Figure 34 The 1H NMR spectrum of A1P1C2T3. Detailed Implementation

[0044] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial sources or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0045] This invention utilizes combinatorial chemistry to construct a multi-aminomidine cationic lipid library, the composition of which is shown in Table 1 below. The chemical structures of each part are represented by A1-A2, P1-P6, C1-C2, and T1-T7, respectively, wherein the general structural formula of A is... (m represents 1 carbon and 2 carbons), the general structural formula of P is P A and P B (where n represents 0, 1, or 2 carbon atoms), the general structural formula of C is: (p represents 1 carbon and 2 carbons), the general structural formula of D is (z represents carbon 6, carbon 7, carbon 8, carbon 9, carbon 10, carbon 11, and carbon 13): Table 1. Schematic diagram of the chemical structure of the polyaminoamidinyl cationic lipid library.

[0046] This lipid library contains 168 polyaminomidine-based cationic lipid molecules. To reduce the time required to screen for the optimal lipid molecules and to study the structure-activity relationship between the chemical structure and delivery efficiency of the aforementioned polyaminomidine-based cationic lipid library, this invention further divides the lipid library into three libraries using a step-by-step screening strategy to investigate the structure-activity relationship of each library. The purpose of the first-level screening is to select the optimal tail structure. A schematic diagram of the cationic lipid libraries selected in the first-level screening is shown in Table 2 below. Table 2. First-level screening of cationic lipid molecules (14 polyaminoamidinyl cationic lipid molecules)

[0047] The synthesized polyaminomidine-containing cationic lipid molecules were prepared into a 10 mM solution. 100 μL of this 10 mM cationic lipid molecule solution was mixed thoroughly with 50 μL of a 10 mM neutral phospholipid (DOPE) solution in an EP tube. Then, 350 μL of a 200 mM sodium acetate buffer solution (pH=5) was added, and the mixture was immediately vortexed for 10 seconds to obtain a two-component lipid nanoparticle solution suitable for verifying delivery efficiency. Unless otherwise specified, the preparation steps for the two-component lipid nanoparticles are the same.

[0048] The delivery efficiency of the lipid nanoparticle solution prepared above was verified in 293T cells. The specific steps are as follows: 1. Add 20 μL of PBS solution to a PCR tube, and then add 1.2 μL of the two-component lipid nanoparticle solution; 2. Dilute EGFP mRNA to a concentration of 220 ng / 10 μL, and add 20 μL of PBS solution to the tube. 1. Prepare a 220 ng / 10 μL mRNA solution, pipette it evenly, and incubate at room temperature for about 30 minutes. 2. Digest 293T cells into a cell suspension, dilute the cells with culture medium to a concentration of 75,000 cells / 100 μL, and after incubation in step 2, add 200 μL of cell suspension to each PCR tube, pipette it evenly, and then add 120 μL of the cell suspension-lipid nanoparticle mixture to each well of a 96-well plate. 3. After 36 hours, add 20 μL of 10 mM Hoechst nuclear staining agent to each well of cells, and stain in the dark at 37°C for about 30 minutes. After successful staining, use a high-content microscope to quantitatively analyze the cell transfection efficiency. The formula for calculating the transfection efficiency is: Unless otherwise specified, the procedures for subsequent cell delivery efficiency verification experiments are the same.

[0049] The mass spectrometry analysis results of the 14 cationic lipid molecules synthesized in the first stage of screening are shown in the figure below. Figures 1-14 As shown, the 1H NMR spectra of A1P4C2T4 and its intermediates C2T4 and P4C2T4 are respectively shown in [reference needed]. Figure 15 ,16 The transfection results of 17.14 cationic lipid molecules in 293T cells are as follows: Figure 18 and 19 As shown in the figure; analysis of the transfection results shows that among the 14 tail chain structures, C1T3, C1T4, C2T3, and C2T4 are the optimal tail chain structures; among the 14 cationic lipid molecules selected in the first stage of screening, A1P1C2T3 is the lipid molecule with the highest transfection efficiency.

[0050] After identifying the four optimal tail structures C1T3, C1T4, C2T3, and C2T4, a second cationic lipid molecule library was constructed to further screen for the optimal benzene ring structure, namely the P structure in Table 1, as shown in Table 3. Table 3. Second-level screening of cationic lipid molecules (24 polyaminoamidinyl cationic lipid molecules)

[0051] The preparation method of lipid nanoparticles and the specific cell transfection experimental steps in the screening process of the second-stage cationic lipid molecules are the same as those described above. The transfection results of the second batch of cationic lipid molecules in mouse hippocampal neurons and mouse microglia are as follows. Figure 20-21 and Figure 22-23 As shown, among the 24 cationic lipid molecules screened in the second stage, A1P4C2T4 showed the highest transfection efficiency, exceeding that of A1P1C2T3. Comprehensive analysis of the transfection results from HT-22 and BV2 cells indicates that p-aminobenzoic acid (P1 structure) and p-aminophenylacetic acid (P4 structure) are the optimal benzene ring structures.

[0052] After identifying the optimal benzene ring structure, in order to further screen for the optimal head amino structure, namely structure A in Table 1, this invention constructed a third cationic lipid molecule library, as shown in Table 4: Table 4. The cationic lipid molecule library selected for the third-level screening (16 polyaminoamidinyl cationic lipid molecules).

[0053] The preparation method of lipid nanoparticles and the specific cell transfection experimental steps in the screening process of the third-stage cationic lipid molecules are the same as those described above. The transfection results of the third batch of cationic lipid molecules in mouse hippocampal neurons and mouse microglia are as follows. Figure 24-25 As shown; comprehensive analysis of the transfection results of HT-22 cells and BV2 cells shows that dimethylaminopropionitrile is the optimal head amino structure (A1).

[0054] Through the above three rounds of cell delivery efficiency verification, this invention discovered the structure-activity relationship of the polyaminomidine lipid library and screened out two candidate polyaminomidine cationic lipid molecules with excellent delivery efficiency in various cell types, namely A1P1C2T3. and A1P4C2T4 .

[0055] Subsequently, further investigations were conducted on the optimal two-component formulation, cytotoxicity, and storage stability of the candidate lipid molecule A1P1C2T3. First, the optimal two-component formulation of A1P1C2T3 was screened, and the screening results (see...) Figure 26 As can be seen, the optimal two-component formulation of A1P1C2T3 is cationic lipid:DOPE = 2:1; subsequently, under the optimal two-component formulation, the optimal transfection dose and cytotoxicity of A1P1C2T3 were evaluated, and the results (see...) Figure 27 and Figure 28 The results showed that the optimal transfection dose of A1P1C2T3 was 1.2 μL / well (96-well plate), and even when the transfection dose was increased to 2.0 μL / well, the cell viability remained above 80%, indicating that A1P1C2T3 has good cell biocompatibility. Finally, the storage stability of the optimal two-component formulation of A1P1C2T3 was evaluated, and the results are shown in […]. Figures 29-31 The results showed that the delivery efficiency of EGFP mRNA from A1P1C2T3 remained almost unchanged after three weeks of storage in a 4°C refrigerator.

[0056] LNP formulations are typically four-component, used because nanoparticles formed from two- or three-component formulations are not necessarily stable. The experimental data provided in this invention (see...) Figure 26-31 This fully demonstrates that the two-component LNP formulation based on A1P1C2T3 ionizable cationic lipid molecules and DOPE molecules in this invention not only exhibits excellent in vitro delivery efficiency in 293T cells and HT22 cells, but also shows no change in delivery efficiency after three weeks of storage in a 4°C refrigerator. This indicates that the two-component LNP formulation based on A1P1C2T3 ionizable cationic lipid molecules and DOPE molecules also possesses good physicochemical stability, suggesting excellent storage stability and making it a promising candidate as a novel two-component or three-component LNP to optimize the pharmacokinetic properties of LNPs.

[0057] The following are examples of A1P1C2T3 and two-component lipid nanoparticles: Example 1 This embodiment provides an ionizable cationic lipid molecule, A1P1C2T3, whose synthetic route, preparation method, and structural formula are as follows:

[0058] S1, 2.4 equivalents of nonylthiol T3, and 1 equivalent of butynediamine C2 (4 mmol) were dissolved in 8 mL of tetrahydrofuran in a 20 mL glass vial. Then, 0.08 equivalents of photoinitiator (2,2-dimethoxy-2-phenylacetophenone, DMPAP) were added. After complete dissolution, the mixture was placed in a UV light generator and reacted for 3 hours. The solvent was rotary evaporated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the intermediate product C2T3. (Petroleum ether:ethyl acetate = 1:1, Rf = 0.2), the yield of this reaction step was 90%. The NMR structure characterization of C2T3 is shown below. Figure 32 ; S2, 1 equivalent of intermediate product C2T3 (2 mmol), and 1.1 equivalent of p-aminobenzoic acid were dissolved in 10 mL of tetrahydrofuran. Then, 1.2 equivalents of 1-hydroxybenzotriazole (HOBt), 1.2 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), and 3 equivalents of N,N-diisopropylethylamine were added sequentially. The mixture was stirred thoroughly and reacted at room temperature for 4 hours. After the UV spot of the starting material p-aminobenzoic acid disappeared as detected by TLC, the reaction solvent was evaporated to stop the reaction, yielding the crude product. The crude product was dissolved in 10 mL of dichloromethane. The organic phase was extracted sequentially with 20 mL of water, 15 mL of saturated sodium bicarbonate solution, 15 mL of saturated ammonium chloride solution, and 15 mL of saturated sodium chloride solution. Finally, the organic phase was collected and the dichloromethane was evaporated to dryness. The crude product was then purified using a silica gel column chromatography to obtain the intermediate product P1C2T3. (Dichloromethane:methanol = 200:1, Rf = 0.2), the yield of this reaction step is 60%. The NMR structure characterization of P1C2T3 is shown below. Figure 33 ; S3, 1 equivalent of the intermediate product P1C2T3 (0.5 mmol), 3.5 equivalents of dimethylaminopropionitrile, and 1.1 equivalents of aluminum chloride were added to a sealed tube. After reacting at 120°C for 1 hour, 10 mL of 0.3 M glacial sodium hydroxide solution was added, and the mixture was stirred thoroughly for 10 min. Then, 10 mL of dichloromethane was added, and the mixture was stirred thoroughly again for 10 min. The dichloromethane was collected and evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain the final product A1P1C2T3. (Dichloromethane:methanol = 10:1, Rf = 0.2), the yield of this reaction step is 30%. The NMR structure characterization of A1P1C2T3 is shown below. Figure 34 .

[0059] Unless otherwise specified, the synthesis steps of other lipid molecules described in this invention are the same as or similar to those described herein.

[0060] Example 2 This embodiment provides the preparation steps of two-component lipid nanoparticles of 14 polyaminomidine-based ionizable cationic lipid molecules A1P1C1T1, A1P1C1T2, A1P1C1T3, A1P1C1T4, A1P1C1T5, A1P1C1T6, A1P1C1T7, A1P1C2T1, A1P1C2T2, A1P1C2T3, A1P1C2T4, A1P1C2T5, A1P1C2T6 and A1P1C2T7, and their transfection steps in 293T cells.

[0061] Preparation steps of the two-component lipid nanoparticles: A 10 mM solution of polyaminomidine cationic lipid molecules was prepared. 100 μL of the 10 mM cationic lipid molecule solution was mixed thoroughly with 50 μL of a 10 mM neutral phospholipid (DOPE) solution in an EP tube. Then, 350 μL of a 200 mM sodium acetate buffer solution (pH=5) was added, and the mixture was immediately vortexed for 10 seconds to obtain the two-component lipid nanoparticle solution suitable for verifying delivery efficiency. Unless otherwise specified, the preparation steps of the two-component lipid nanoparticles described in this invention are the same or similar.

[0062] The delivery efficiency of the two-component lipid nanoparticles prepared above was verified in 293T cells. The specific steps are as follows: 1. Add 20 μL of PBS solution to a PCR tube, then add 2.4 μL of the two-component lipid nanoparticle solution; 2. Dilute EGFP mRNA to a concentration of 220 ng / 10 μL, and add 20 μL of PBS solution to the tube. 1. Prepare a 220 ng / 10 μL mRNA solution, pipette it evenly, and incubate at room temperature for approximately 30 minutes. 2. Digest 293T cells into a cell suspension, dilute the cells with culture medium to a concentration of 75,000 cells / 100 μL, and after the incubation step in step 2, add 200 μL of cell suspension to each PCR tube, gently pipette it evenly, and then add 120 μL of the cell suspension-lipid nanoparticle mixture to each well of a 96-well plate. 3. After 36 hours, add 20 μL of 10 mM Hoechst nuclear staining agent to each well of cells and stain in the dark at 37°C for approximately 30 minutes. After successful staining, use a high-content microscope to quantitatively analyze the cell transfection efficiency. The formula for calculating the transfection efficiency is: The results and analysis of 293T cell transfection with 14 lipid molecules are described above. Figure 18 And its analysis. Unless otherwise specified, the steps of the cell delivery efficiency verification experiment described in this invention are the same as or similar to those described herein.

[0063] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A cationic lipid compound, characterized in that, Its structural formula is shown in Formula I: Formula I; Wherein, m, n, and p are independently selected from integers 1~8, 0~8, and 1~8, respectively; The X 1 It is an amide bond or an ester bond; The R 1 The optional substituted (C1-C6) alkylamine group, the optional substituted bis(C1-C6) alkylamine group, the optional substituted aromatic amine group, or the optional substituted nitrogen heterocyclic group; wherein the nitrogen atom of the optionally substituted nitrogen heterocyclic group is... Connected; The R 2 Selected from (C5-C) which are optionally substituted 25 )alkyl, optionally substituted (C5-C 25 alkenyl; The R 3 Selected from halogen, cyano, isocyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 carbocyclic, C6-C 10 Aryl; k is an integer selected from 0 to 4; when k ≥ 2 are different, multiple R 3 They can be the same or different; The substitution refers to the presence of a substituent at one or more substituted positions of the group, wherein the substituent is independently halogen, cyano, isocyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 carbocyclic, C6-C 10 Aryl, C5-C 10 Mixed aromatic compounds.

2. The cationic lipid compound according to claim 1, characterized in that, The m is selected from 1, 2, or 3; And / or, n is selected from 0, 1 or 2; And / or, p is selected from 1, 2 or 3.

3. The cationic lipid compound according to claim 1, characterized in that, The X 1 Selected from: , , , The dashed lines represent the connection points of the functional groups. The left side of each depicted structure is... Combine; The R 3 Independently selected from hydrogen or optionally substituted (C1-C6) alkyl groups.

4. The cationic lipid compound according to claim 1, characterized in that, The R 1 Selected from methylamino, ethylamino, propylamino, dimethylamino, diethylamino, dipropylamino, aniline, naphthylamino, anthraceneamino, pyridyl, and pyrrole.

5. The cationic lipid compound according to claim 1, characterized in that, The R 2 Selected from (C5-C) which are optionally substituted 15 )alkyl, wherein the substituent is independently halogen or C1-C6 alkyl.

6. A method for preparing the cationic lipid compound according to any one of claims 1 to 5, characterized in that, The process includes the following steps: reacting compound F with compound A to obtain compound I; The structural formulas of compound F and compound A are shown below: Formula F; Formula A; Wherein, m, n, p, k, X 1 R 1 R 2 R 3 As described in any one of claims 1 to 5.

7. A lipid composition, characterized in that, It comprises the cationic lipid compound as described in any one of claims 1 to 5, and further comprises at least one of the following lipids: (i) one or more non-cationic lipids; (ii) One or more cholesterol-based lipids; (iii) One or more PEG-modified lipids.

8. The lipid composition according to claim 7, characterized in that, The lipid composition is microparticles, nanoparticles, liposomes, lipid nanoparticles, or microbubbles.

9. The use of the cationic lipid compound according to any one of claims 1 to 5, or the lipid composition according to claim 7 or 8, in the delivery of bioactive substances.

10. The application according to claim 9, characterized in that, The bioactive substances are small molecule compounds, nucleic acids, proteins, peptides, metals, isotopically labeled compounds, and vaccines.

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